Fig 1.
Phylogenetic relations of L. salmonis nAChR subunits.
Consensus phylogram constructed using full-length nAChRs subunits (amino acid sequences) from Lepeophtheirus salmonis (Lsa), Apis melifera (Ama), Drosophila melanogaster (Dme) and Aedes aegypti (Aae). Branch labels indicate the consensus support in percent. The tree was constructed using a maximum likelihood approach (PhyML 3.0) and a bootstrap with 1000 iterations. The scale bar represents 0.3 substitutions per site. Black diamonds indicate β subunits.
Fig 2.
Determination of the minimum number of receptor subunits and ancillary proteins required for efficient expression of Lsa-nAChR.
A) Co-injection (equimolar ratio) of six nAChR subunits (black squares) and the three ancillary proteins (grey squares; AP) yields strong currents with 100 μM ACh (1st column). Injection of all alpha (α) or both beta (β) receptor subunits and APs failed to produce any measurable current (column 2 and 3). The same was true when APs were removed (column 4) or when Lsa-ric-3 or Lsa-unc-50 and Lsa-74 where combined with all the receptor subunits (column 5 and 6 respectively). B) Each individual nAChR-α subunit was co-injected with both nAChR-β subunit and APs (column 7 to 10). Only the Lsa-nAChR-α3 subunit was able to form a functional receptor gated by 100 μM ACh (column 9). C) The combination of Lsa-nAChR-α1 and Lsa-nAChR-α2 with both β receptor subunits and APs in oocytes challenged with 100 μM ACh also yields currents (column 11) while removing Lsa-nAChR-β2 (column 12) or Lsa-nAChR-β1 (column 13) from the injection mix almost or completely abolished the current. Combination of Lsa-nAChR-α1 or Lsa-nAChR-α2 with Lsa-nAChR-α7 with both β receptor subunits and APs (column 14 and 15 respectively) did not yield any current. Removing any of the β receptor subunit from the injection mix containing Lsa-nAChR-α3 (column 16 and 17) abolished the current in both cases. Grey points represent individual oocyte recording for each condition, black horizontal bar the median as well as the interquartile range. Numbers above the plot indicate the number of oocytes recorded for each condition. Y-axis is discontinuous to make the smaller currents recorded from the column 12 visible.
Fig 3.
Hypothetical stoichiometry, current traces and dose response curves of L. salmonis nAChRs.
Hypothetical 3D model of a Lsa-nAChR (A). Subunits composition as well as current traces and averaged dose response curves from a cumulative exposure to increasing dosage of acetylcholine (in μM) for Lsa-nAChR-1 (B, D and F) and Lsa-nAChR-2 (C, E and G) are shown. Individual curves were standardized to the fitted maximal current amplitude and subsequently averaged. Mean ± SEM of experiments carried out with at least six oocytes from two batches is shown. For both receptors, the exact stoichiometry is not known yet and the precise relative positioning of each subunit remains to be determined. The question marks (B and C) represent copies of one of the subunits already indicated. At high ACh concentration (300 μM; D and E), an open channel block is visible (smaller currents than at 100 μM) as well as bounce back current when the agonist is removed (relief from the agonist block).
Fig 4.
Effect of two different α:β ratio on Lsa-nAChR1 and Lsa-nAChR2 sensitivity to acetylcholine.
Typical current traces and averaged dose response curves from a cumulative exposure to increasing dosage of ACh (in μM) for Lsa-nAChR-1 (A, B and C) and Lsa-nAChR-2 (D, E and F). The α:β ratio used for each experiment is indicated below the traces. Individual curves were standardized to the fitted maximal current amplitude and subsequently averaged. Mean ± SEM of experiments carried out with at least two oocytes from two batches is shown.
Table 1.
Characterization of two different compositions of Lsa-nAChR1 and Lsa-nAChR2.
The different combinations tested had no significant effect on the ACh EC50 value on Lsa-nAChR1 while a statistically significant lower ACh EC50 value was recorded with the α/β = 10:1 ratio for Lsa-nAChR2. "n = " refers to the number of individual cells evaluated.
Fig 5.
Effect of two different α:β ratio on Lsa-nAChR1 and Lsa-nAChR2 sensitivity to sazetidin A (Saz A) and dihydro-β-erythroidine (DHbE).
Typical current traces obtained with 10 μM Saz A (A and C) or 10 μM DHbE (B and D) obtained from oocytes expressing two different ratios of α:β Lsa-nAChR1 (A and B) or Lsa-nAChR2 (C and D) are shown. The bars indicate the time period of ACh (black line), Saz A (interrupted line), or DHbE (grey line). The traces obtained with Saz A are also represented with a smaller scale to allow a better visualization of the peak currents recorded.
Fig 6.
Agonist screening on Lsa-nAChR1 and Lsa-nAChR2.
Typical current traces obtained with different agonists tested on Lsa-nAChR1 (A) or Lsa-nAChR2 (B). ACh (1) was tested at 100 μM) while all other compounds (2 to 13) were tested at 1 μM. 1) acetylcholine 100 μM; 2) azamethiphos; 3) emamectin; 4) nitenpyram; 5) deltamethrine; 6) thiacloprid; 7) clothianidin; 8) imidacloprid; 9) thiamethoxam; 10) acetamiprid; 11) dinotefuran; 12) cypermethrin; 13) acetylcholine 1 μM.
Table 2.
Comparison of sensitivities of Lsa-nAChR1 and Lsa-nAChR2 expressed in Xenopus oocytes to neonicotinoids and non-nicotinoid compounds.
The compounds were tested on Xenopus laevis oocytes expressing these Lsa-nAChRs injected at a 1:1 (α:β) ratio. The table also gives results from an in-vivo assay in which salmon lice were exposed to seven neonicotinoids at 1 mg/L for 24 h. "n = " refers to the number of individual cells evaluated.